Studies in both humans and experimental models indicate that T cells play an important role in antitumor responses. Identification of MHC class I-restricted melanoma antigens has provided new opportunities to develop antigen-specific antitumor immunity. However, the clinical responses elicited by such vaccines have been weak and transient, suggesting the need for CD4+ T cells to generate optimal antitumor immunity. Despite the recognized role of CD4+ T (helper) cells in antitumor immunity, relatively little is known about the MHC class II-restricted antigens recognized by tumor-specific CD4+ T cells, posing a major obstacle to the development of more effective cancer vaccines. The goal of this proposal is to identify and test MHC class II-restricted melanoma peptides that are presented on the cell surface for CD4+ T cell recognition in the context of HLA-DR3, and -DR13 molecules, which are expressed in more than 45% of the general population. The underlying hypothesis is that vaccination with MHC class II antigens, in combination with an effective MHC class I peptide, will stimulate tumor-specific CD4+ T cells, leading to more potent antitumor immunity than could be obtained using either peptide alone. Four specific research aims are proposed to test this prediction. Aim 1 will seek to identify new class II-restricted peptides through the use of recently established tumor reactive CD4+ T cell lines/clones and a novel cloning technology. The ability of these and previously identified MHC class II peptides to induce CD4+ T cells will then be enhanced by modifying key residues in T-helper peptides and delivering them into dendritic cells (DCs) (Aim 2). The immunogenicity of the most promising peptide candidates emerging from Aim 2 will be further evaluated by comparing their ability to elicit CD4+ T cell response from patient-derived peripheral blood mononuclear cells (PBMCs) to responses in an HLA-DR transgenic mouse model, using peptide-loaded DCs (Aim 3). Finally, in Aim 4 the immunogenicity, as well as safety and efficacy, of the candidate peptides will be subjected to clinical testing in melanoma patients, and to evaluation of the immunotherapeutic potential of a combined MHC class I and II peptide vaccine. The applicants' record of success in the generation and testing of immunogenic tumor antigens, together with experience in clinical trials, bode well for further advances in this important area of cancer immunotherapy.